Method for producing arbuscular mycorrhizal fungus

Abstract

To provide a novel method that enables efficient production of an arbuscular mycorrhizal fungus.SOLUTION: A method for producing an arbuscular mycorrhizal fungus, the method comprising a culturing step of culturing an arbuscular mycorrhizal fungus and a first plant infected with the arbuscular mycorrhizal fungus in the presence of a porous support to propagate the arbuscular mycorrhizal fungus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing arbuscular mycorrhizal fungi, a method for producing plants infected with arbuscular mycorrhizal fungi, a method for cultivating plants, and the like. [Background technology]

[0002] Approximately 80% of terrestrial plants are infected with arbuscular mycorrhizal fungi, which provide host plants with water and nutrients such as phosphate, nitrogen, and zinc, contributing to plant growth, development, drought stress tolerance, and resistance to plant pathogens, thereby significantly improving plant growth.

[0003] When arbuscular mycorrhizal fungi infect plant roots, their hyphae penetrate the plant's epidermal cells and branch repeatedly to form arbuscules, which are structures for nutrient exchange. In the soil, the hyphae extend into areas that plant roots cannot reach and into soil pores, providing nutrients such as phosphorus, nitrogen, sulfur, potassium, calcium, copper, and zinc from the soil to the host plant.

[0004] Because arbuscular mycorrhizal fungi can significantly improve plant growth based on the above-mentioned effects, they are expected to replace chemical fertilizers (especially phosphorus fertilization), and their use in agriculture is being promoted.

[0005] However, it is currently difficult to efficiently produce arbuscular mycorrhizal fungi. Patent Document 1 discloses a method for growing arbuscular mycorrhizal fungi by culturing them together with plant roots. This method has problems such as a low spore production rate and a long culture period of five months, making it difficult to mass-produce arbuscular mycorrhizal fungi in a short period of time.

[0006] Furthermore, methods for promoting plant cultivation using propagated arbuscular mycorrhizal fungi are currently insufficient. Currently, fertilizers containing arbuscular mycorrhizal fungi are commercially available, but even if plants are grown in soil to which this fertilizer has been added, the expected effects are not fully achieved. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication 2016-528906 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a new method for efficiently producing arbuscular mycorrhizal fungi. [Means for solving the problem]

[0009] In order to find a method for efficiently growing arbuscular mycorrhizal fungi, the present inventors cultivated arbuscular mycorrhizal fungi together with flax hairy roots in liquid media in which various supports were immersed.

[0010] As a result, we found that arbuscular mycorrhizal fungi can grow extremely efficiently in a liquid medium immersed in a porous support with pores on the surface. According to the method of the present invention, the amount of spores produced per liter of culture medium is approximately five times higher than that of the conventional method disclosed in JP-A-2016-528906, and the time required for spore production can also be significantly reduced.

[0011] Furthermore, arbuscular mycorrhizal fungal hyphae were observed adhering to the surface of the porous support immersed in liquid medium, and the hyphae were also observed penetrating into the pores. These results suggest that arbuscular mycorrhizal fungal hyphae can grow efficiently by using the surface and pores of the porous support as a foothold.

[0012] On the other hand, when other supports without pores on the surface were immersed in liquid medium, the growth rate of arbuscular mycorrhizal fungi was low and sufficient growth was not achieved.

[0013] The present invention is based on the above findings and provides the following. (1) A method for producing arbuscular mycorrhizal fungi, the method comprising a culturing step of culturing arbuscular mycorrhizal fungi and a first plant to be infected with the arbuscular mycorrhizal fungi in the presence of a porous support to proliferate the arbuscular mycorrhizal fungi. (2) The method according to (1), wherein in the culturing step, the hyphae of the arbuscular mycorrhizal fungi attach to the porous support and / or penetrate into the pores of the porous support. (3) The method according to (1), wherein the culturing step cultures the roots of the first plant. (4) The method according to (3), wherein the roots are hairy roots. (5) The method according to (1), wherein the first plant is other than the Brassicaceae and Chenopodiaceae families. (6) The method according to (1), wherein the culture is an axenic culture. (7) The method according to (1), wherein the culture is a liquid culture. (8) The method according to (7), wherein the specific gravity of the porous support is greater than the specific gravity of the culture solution used for the liquid culture. (9) The method according to (1), wherein the porous support has an average pore diameter of 100 nm or more and 1 μm or less. (10) The method according to any one of (1) to (9), wherein the porous support is made of expanded brick. (11) The method according to (1), further comprising a spore recovery step of recovering the arbuscular mycorrhizal fungal spores obtained after the culture step. (12) A method for producing a plant infected with an arbuscular mycorrhizal fungus, the method comprising an infection step of culturing an arbuscular mycorrhizal fungus and a plant to be infected with the arbuscular mycorrhizal fungus in the presence of a porous support to infect the plant with the arbuscular mycorrhizal fungus. (13) A method for producing a plant infected with an arbuscular mycorrhizal fungus, the method comprising an infection step of infecting a second plant with the arbuscular mycorrhizal fungus produced by the method described in (1). (14) A method for cultivating a plant, comprising a cultivation step of cultivating a second plant to be infected with the arbuscular mycorrhizal fungus together with the arbuscular mycorrhizal fungus produced by the method described in (1). (15) The method according to (14), wherein the cultivation step is soil cultivation or hydroponic cultivation. (16) The method according to (15), further comprising an addition step of adding the arbuscular mycorrhizal fungi to the soil used for the soil cultivation or the hydroponic solution used for the hydroponic cultivation before the cultivation step. (17) The method according to (14), further comprising an infection step of infecting the second plant with the arbuscular mycorrhizal fungus before the cultivation step. (18) The method according to any one of (13) to (17), wherein the second plant is other than the Brassicaceae and Chenopodiaceae families. (19) A porous support to which hyphae and / or spores of arbuscular mycorrhizal fungi are attached. (20) Hyphae and / or spores of arbuscular mycorrhizal fungi attached to a porous support. (21) A plant cultivation set comprising the porous support described in (19), the arbuscular mycorrhizal fungus, a plant infected with the arbuscular mycorrhizal fungus, and soil or a hydroponic solution for cultivating the plant. [Effects of the Invention]

[0014] According to the present invention, a new method for efficiently producing arbuscular mycorrhizal fungi is provided. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the growth of flax hairy roots cultured in the presence and absence of hydroball (I). [Figure 2]Photographs of flax hairy roots cultured in the presence and absence of Hydroball (I) taken from a liquid medium and taken under a stereomicroscope, with the arrowheads indicating high-density spore growth. [Figure 3] 1 shows spores collected from flax hairy roots cultured in the presence and absence of hydroball (I), with arrowheads indicating spores. [Figure 4] FIG. 1 shows the results of quantifying the number of spores recovered from flax hairy roots cultured in the presence and absence of hydroball (I). [Figure 5] FIG. 1 shows that spores grown in the presence of Hydroball (I) have the ability to germinate. [Figure 6] This is a photograph taken under a stereomicroscope of hydroballs (I) and flax hairy roots removed from the liquid medium during liquid culture in Example 1. The photograph on the right shows an enlarged view of the area within the frame in the photograph on the left. [Figure 7] FIG. 1 is a photograph showing the surface structure of Hydroball (I) taken under a microscope. [Figure 8] FIG. 1 is a photograph showing the surface structure of Hydroball (II) taken under a microscope. [Figure 9] FIG. 1 shows a photograph of the surface structure of Jiffy Seven PLA taken under a microscope. [Figure 10] FIG. 1 is a photograph of the surface structure of rice husks taken under a microscope. [Figure 11] FIG. 1 is a photograph of the surface structure of Mikawa silica sand taken under a microscope. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1. Arbuscular mycorrhizal fungi production method (AM fungi production method) Overview A first aspect of the present invention is a method for producing arbuscular mycorrhizal fungi (hereinafter sometimes abbreviated as "AM fungi production method"). The AM fungi production method of this aspect includes, as an essential step, a culture step in which arbuscular mycorrhizal fungi and a plant are cultured in the presence of a porous support, and can extremely efficiently grow arbuscular mycorrhizal fungi. In the culture step of the AM fungi production method of this aspect, the porous support provides a scaffold for the hyphae of the arbuscular mycorrhizal fungi, thereby promoting the growth of the arbuscular mycorrhizal fungi.

[0017] 1-2.Definition of Terms Terms frequently used in this specification are defined below. As used herein, "arbuscular mycorrhizal fungi" is a general term for filamentous fungi that are ubiquitous in soil and can coexist symbiotically with approximately 80% of terrestrial plants. In this specification, they are often referred to as "AM fungi." AM fungi primarily receive glucose from plants, but instead collect moisture and nutrients such as phosphate from the soil and provide them to the host plant. Upon infection, AM fungi form characteristic arbuscule structures within the roots of the host plant, and phosphate and other nutrients are supplied to the plant through these arbuscule structures. AM fungi produce spores, which are extremely large for fungi, measuring 0.1 to 0.5 mm in size, on external hyphae that develop outside the roots. AM fungal spores in the soil germinate and grow hyphae when given the appropriate temperature and moisture.

[0018] Currently, there are over 300 known species of AM fungi, including those in the orders Archaeosporales, Glomerales, Diversisporales, and Paraglomerales, but the AM fungi referred to herein are not particularly limited and may be any of them. Examples of the order Archaeosporales include the family Ambisporaceae (e.g., Ambispora fennica), Archaeosporaceae (e.g., Acaulospora trappei), Geosiphonaceae (e.g., Geosiphon pyriformis), and family Polonasporaceae (e.g., Acaulospora polinica). Examples of the Glomerales include the family Glomeraceae (e.g. Rhizophagus diaphanum, Glomus minutum, Epigeocarpum crypticum, Endogone mosseae, Funneliglomus sanmartinense, Glomus macrocarpum, Halonatospora pansihalos, Glomus bistratum, Dominikia litorea, Microkamienskia) peruviana, Nanoglomus plukenetiae, Glomus diaphanum, Dominikia emiratia, Rhizophagus populinus, Sclerocarpum amazonicum, Sclerocystis coremioides, Glomus constrictum, Rhizophagus neocaledonicus) and Claroideoglomeraceae (e.g. Glomus claroideum).Examples of Diversisporales include Acaulosporaceae (e.g., Acaulospora laevis), Diversisporaceae (e.g., Corymbiglomus corymbiforme, Glomus spurcum, Diversispora omaniana, Otospora bareai, Glomus megalocarpum, Glomus tortuosum, Entrophospora nevadensis), Pacisporaceae (e.g., Glomus scintillans), Sacculosporaceae (e.g., Entrophospora baltica), and Gigasporaceae (e.g., Bulbospora minima, Gigaspora gilmorei, Gigaspora nigra, Racocetra intraornata, Endogone gigantea, Paradentiscutata Examples of the Paraglomerales include the Paraglomeraceae (e.g., Paraglomus majewskii, Paraglomus occultum) and the Pervetustaceae (e.g., Pervetustus simplex). Further examples of AM fungi include the Entrophosporales (e.g., the Entrophosporaceae, e.g., Glomus infrequens).

[0019] As used herein, the term "hyphae" refers to a filamentous structure formed by multicellular fungi. In this specification, the term generally refers to the hyphae of AM fungi. The hyphae of AM fungi extend through the soil using nutrients stored in spores, and when they encounter the roots of a host plant, they penetrate the roots and form the above-mentioned arbuscule inside the roots.

[0020] As used herein, the term "support" refers to something that is placed in soil, hydroponic solution, or culture solution to aid in plant cultivation. The material of the support is not particularly limited, and examples other than the porous supports described below include Jiffy Seven, rice husks, Mikawa silica sand, synthetic resins such as plastic, glass, metal, etc. The shape of the support is not particularly limited, but a shape that has a large contact surface area with the soil, hydroponic solution, or culture solution is preferred, and may be, for example, spherical, cubic, or cylindrical. The size of the support is, for example, 1 mm 3 More than 100cm 3 Below, 10mm 3 More than 50cm 3 Below, 100mm 3 More than 20cm 3 Below, 1cm 3 More than 10cm 3 Less than or equal to 2cm 3 More than 5cm 3 The length (diameter or length of one side) of the support may be, for example, 0.1 mm to 10 cm, 1 mm to 5 cm, 2 mm to 4 cm, 5 mm to 3 cm, or 1 cm to 2 cm, and is preferably 2 mm to 1 cm, 3 mm to 8 mm, or 4 mm to 6 mm.

[0021] In this specification, the term "porous support" refers to the support described above that has a large number of pores at least in its surface layer. Examples of porous supports include foamed brick, activated carbon, bamboo charcoal, ceramics, zeolite, silica gel, pumice, porous glass, alumina, polyurethane, etc., with foamed brick being particularly preferred.

[0022] In this specification, "expanded brick" refers to a solid material made by baking clay at high temperatures to foam it. Exploded bricks are also called hydroballs. Exploded bricks are also used as supports for hydroponic cultivation, but no method has been known to date for cultivating plants infected with AM fungi in the presence of expansive bricks.

[0023] As used herein, "hydroponic cultivation" refers to a form of cultivation in which all or part of the roots of a cultivated plant are immersed in a hydroponic solution. "Hydroponic solution" refers to a liquid medium or liquid fertilizer used in hydroponic cultivation. The hydroponic solution used in this plant cultivation method may be any hydroponic solution known in the art. An example of a hydroponic solution is a liquid medium containing 10% Murashige and Skoog medium (MS medium), 1% sucrose, and vitamins. The amount of hydroponic solution may be selected appropriately depending on the volume of the cultivation container, and is, for example, from 1 mL to 100 L, from 10 mL to 10 L, or from 100 mL to 1 L. Hydroponic solutions are also commercially available from gardening equipment manufacturers, and these can also be used.

[0024] As used herein, "soil cultivation" refers to cultivation using natural or artificial soil. The amount of soil used for cultivation may be selected appropriately depending on the volume of the cultivation container, and may be, for example, from 1 mL to 100 L, from 10 mL to 10 L, or from 100 mL to 1 L. Cultivation soil is also commercially available from gardening equipment manufacturers, and such soil may also be used.

[0025] As used herein, the term "cultivation container" refers to a container for holding soil, hydroponic solution, or culture solution used for cultivating plants.

[0026] As used herein, the term "plant" refers to any plant with which AM fungi can form mycorrhizae. Specifically, it refers to any plant other than those in the Brassicaceae and Chenopodiaceae families, and may be either angiosperms or gymnosperms. Angiosperms also include both dicotyledonous and monocotyledonous plants. For example, plants include those important in agriculture, particularly in the seed and floriculture industries, such as crop plants such as cereals, flowers, vegetables, and fruits. Specific examples of dicotyledonous plants include species belonging to the Linaceae family (e.g., flax), species belonging to the Fabaceae family (e.g., soybean, peanut, pea, kidney bean, adzuki bean, broad bean, sweet pea), species belonging to the Solanaceae family (e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, petunia), species belonging to the Rosaceae family (e.g., strawberry, rose, apple, pear, peach, loquat, almond, plum, plum, cherry), and species belonging to the Orchidaceae family (e.g., Cymbidium, Phalaenopsis, Examples of monocotyledonous plants include species belonging to the family Liliaceae (e.g., lilies, tulips, hyacinths, muscari, leeks, onions, garlic), family Rutaceae (e.g., mandarins, oranges, grapefruits, lemons, and yuzu), family Vitaceae (e.g., grapes), family Asteraceae (e.g., lettuce, chrysanthemums, dahlias, marigolds, and sunflowers), family Caryophyllaceae (e.g., carnations and baby's breath), and family Theaceae (e.g., camellia and tea plants). Furthermore, examples of monocotyledonous plants include species belonging to the family Poaceae (e.g., rice, wheat, barley, corn, sugarcane, sorghum, and sorghum).

[0027] As used herein, the term "all" of a plant refers to all regions constituting a living plant. Furthermore, the term "part" of a plant refers to a portion of a living plant, specifically an organ (e.g., roots, stems, leaves, flowers, fruits, epidermis, or a combination thereof, or pollen, egg cells, seeds, etc.), a tissue or part thereof consisting of a group of morphologically and / or functionally differentiated cells, or a cell.

[0028] 1-3. Method The method for producing AM fungi of the present invention includes a culturing step as an essential step and a spore recovery step as an optional step.

[0029] (Culture process) In this embodiment, the "cultivation step" is a step of culturing the AM fungus and the first plant to be infected with the AM fungus in the presence of a porous support to allow the AM fungus to grow.

[0030] As used herein, the term "first plant" (sometimes simply referred to as "plant" herein) refers to a plant that is cultured with AM fungi for the purpose of propagating the AM fungi. The first plant may be any plant with which AM fungi can form mycorrhizae, such as any plant outside the Brassicaceae and Chenopodiaceae families, and specific examples are as described above for "plant." Preferred examples include flax (e.g., flax hairy roots), chicory (e.g., chicory hairy roots), and carrot (e.g., carrot hairy roots), which can be infected with AM fungi and are easy to culture in liquid.

[0031] As used herein, "in the presence of a porous support" refers to placing the porous support in soil, hydroponic solution, or culture solution so that the hyphae of the AM fungus that has infected the first plant can attach. More specifically, the porous support may be placed near the roots of the first plant. For example, the porous support may be contained in the soil, hydroponic solution, or culture solution used for the culture in this step, or the inner surface of the cultivation container may be made of a porous support. Furthermore, the porous support may be immersed or floating in the hydroponic solution or culture solution, but it is more preferable that it be immersed to facilitate contact with the plant roots.

[0032] In this step, the hyphae of the AM fungus adhere to the porous support, preferably penetrating into the pores of the porous support. As used herein, the phrase "the hyphae penetrate into the pores of the porous support" means that the hyphae have reached the interior of the pores of the porous support, preferably that the hyphae have attached to the surface inside the pores. The attachment of the hyphae of the AM fungus to the porous support provides a foothold for the hyphae, which can promote the growth of the AM fungus and the growth of the first plant.

[0033] In the present invention, the material of the porous support is not limited as long as the mycelia of AM fungi can adhere to its surface. Examples include foam brick, activated carbon, ceramics, zeolite, silica gel, pumice, porous glass, alumina, and polyurethane, with foam brick being preferred. Foam brick is a porous support obtained by baking clay at high temperatures to foam it, as described above, and those containing silicon oxide and aluminum oxide as the main components are particularly preferred. The cumulative pore volume of the porous support is, for example, 0.1 mL / g to 5.0 mL / g, 0.2 mL / g to 4.0 mL / g, 0.3 mL / g to 3.0 mL / g, 0.5 mL / g to 2.0 mL / g, 0.6 mL / g to 1.5 mL / g, or 0.7 mL / g to 1.2 mL / g, preferably 0.8 mL / g to 1.1 mL / g or 0.85 mL / g to 1.05 mL / g. The cumulative pore specific surface area of ​​the porous support is, for example, 10 m 2 / g or more 100m 2 / g or less, 12m 2 / g or more 50m 2 / g or less, or 15m 2 / g or more 40m 2 / g or less, preferably 20m 2 / g or more 30m 2 / g or less, 22m 2 / g or more 28m 2 / g or less, or 24m 2 / g or more 26m 2 / g or less. The average pore diameter of the porous support is, for example, 0.001 μm or more and 10 μm or less, 0.01 μm or more and 5 μm or less, or 0.02 μm or more and 1 μm or less, preferably 0.05 μm or more and 0.5 μm or less, and more preferably 0.1 μm or more and 0.2 μm or less. In the case of soil cultivation, the specific gravity of the porous support is not particularly important, but in the case of hydroponic cultivation or liquid culture, since the support is immersed in the hydroponic solution or culture solution, the specific gravity of the porous support is preferably greater than the specific gravity of the hydroponic solution or culture solution, for example, 1 g / cm 3 However, even if the porous support has a low specific gravity, such as pumice, it can be immersed in the hydroponic solution or culture solution by combining it with a substance having a high specific gravity, so the specific gravity is not an issue.

[0034] In one embodiment, the porous support is made of expanded brick having a diameter of 2 mm to 1 cm or 4 mm to 8 mm, and the shape of the porous support is, for example, spherical and / or cylindrical. The average pore diameter of the porous support is, for example, 0.12 μm to 0.20 μm, preferably 0.13 μm to 0.18 μm. The cumulative pore specific surface area of ​​the porous support is, for example, 10 m 2 / g or more 100m 2 / g or less, preferably 20m 2 / g or more 30m 2 / g or less, 22m 2 / g or more 28m 2 / g or less, or 24m 2 / g or more 26m 2 / g or less.

[0035] The culture conditions in this step are not particularly limited as long as they allow the cultivation of the AM fungus and the first plant. For example, the culture can be performed under conditions similar to those of general AM fungal culture methods. The culture temperature is typically between 15°C and 40°C, between 20°C and 35°C, or between 25°C and 30°C. The culture period is, for example, between one day and one year, between one week and six months, between two weeks and five months, between one month and four months, or between two months and three months. The culture may be, for example, soil culture, hydroponic culture, or liquid culture. Furthermore, the cultivation conditions, such as the light-dark period (light exposure time and dark period), climatic conditions including temperature and humidity, and growth period, may be conditions known in the art depending on the type of first plant.

[0036] In one embodiment of this step, in addition to the porous support, an additional support may also be used for culture. Examples of the additional support include inorganic materials such as urethane, rock wool, sand, gravel, vermiculite, and perlite, unhumic cellulose such as sawdust, rice husks, coconut shells, and bark chips, and natural organic materials such as agar, or a combination thereof.

[0037] The method for culturing the AM fungus and the first plant in this step is not limited as long as the AM fungus can infect the first plant during culturing. For example, the first plant infected with the AM fungus may be cultured in the presence of a porous support. Alternatively, the first plant may be cultured in the presence of a porous support to which AM fungal hyphae and / or spores are attached. Furthermore, the first plant may be cultured in the presence of a porous support using soil or a hydroponic solution to which AM fungus has been added.

[0038] The culture in this step may be soil culture, hydroponic culture, or liquid culture, but hydroponic culture or liquid culture is more preferred because soil culture requires the separation of spores from the soil. Furthermore, since hydroponic culture or liquid culture uses a hydroponic solution or liquid medium, it is possible to replace, change, or add the medium during culture. In hydroponic culture or liquid culture, it is sufficient if a porous support is included in part of the hydroponic solution or liquid medium, but it is preferable that the hydroponic solution or liquid medium is filled with the porous support.

[0039] In one embodiment, the culture in this step is a sterile culture. As used herein, "sterile culture" means culturing in an environment in which microorganisms other than AM fungi have been killed or removed and are substantially free of such microorganisms. For example, in a culture environment using sterile soil, hydroponic solution, or liquid medium, the sterility assurance level (SAL; SAL refers to the maximum probability of survival of contaminating bacteria in a product sterilized by an appropriate sterilization process) required internationally in medical settings is 10% or less, excluding AM fungi. -6 By carrying out this step aseptic culture, only the AM fungi can be grown, which has the advantage of making it easier to collect the hyphae and / or spores of the AM fungi.

[0040] In one embodiment, the process involves culturing roots of the first plant. The roots cultured may be, for example, hairy roots.

[0041] The AM fungi grown by the culture in this step may be collected from the arbores of the roots of the first plant. Furthermore, the mycelia and / or spores may be collected from the soil or hydroponic solution, or from the surface of the roots and / or the porous support. Alternatively, the mycelia and / or spores may be collected together with the porous support to which they are attached.

[0042] (Spore recovery process) In this embodiment, the "spore collection step" refers to a step of collecting AM fungal spores obtained after the culture step. Any known method can be used to collect the spores in this step. Examples of known methods include a wet collection method and a dry collection method.

[0043] As used herein, the term "wet recovery method" refers to a method for recovering spores by filtering a liquid such as a culture solution containing spores using a screen mesh, filter cloth, or the like. For example, a method may be used in which the culture after cultivation is dispersed in a buffer solution or physiological saline, and the resulting suspension is separated into a culture residue and a spore dispersion using filter paper, filter cloth, a sieve, or the like. Another method involves mixing the culture with a non-aqueous liquid such as hydrocarbon oil, fatty acid ester oil, or silicone oil, and removing the culture residue from the resulting mixture by sedimentation or fractionation using a filter cloth, wire mesh, or the like.

[0044] As used herein, the term "dry collection method" refers to a method for collecting spores from non-liquid samples using a dry sieve or by vibration or air classification, etc. For example, a method may be used in which a culture such as soil containing spores is crushed and sieved.

[0045] 1-4.Effects According to the AM fungus production method of the present invention, the growth efficiency of AM fungi and plants is higher than when they are cultured in the absence of a porous support or in the presence of a support other than a porous support. Furthermore, when AM fungi and plants are cultured in the presence of foamed brick, the AM fungi grow with particularly high efficiency compared to when other porous supports such as activated carbon are used. Therefore, foamed brick is particularly preferred among porous supports.

[0046] 2. Method for producing arbuscular mycorrhizal fungi-infected plants (method for producing AM fungi-infected plants) 2-1. Overview A second aspect of the present invention is a method for producing a plant infected with AM fungi (hereinafter sometimes abbreviated as "method for producing an AM fungus-infected plant").

[0047] 2-2. Method The method for producing an AM fungus-infected plant of the present invention includes an infection step as an essential step and a cultivation step as an optional step.

[0048] (infection process) In this embodiment, the "infection step" is a step of infecting a second plant with an AM fungus.

[0049] As used herein, the term "second plant" (sometimes simply referred to as "plant" herein) refers to a plant whose growth, development, drought stress tolerance, and / or resistance to plant pathogens is improved by infection with AM fungi. As used herein, the second plant is distinguished from the first plant described above, which is used for the purpose of propagating AM fungi, and may be the same species as the first plant or a different species. The second plant may be any plant with which AM fungi can form mycorrhizae, such as any plant other than the Brassicaceae and Chenopodiaceae families, and may be either angiosperms or gymnosperms. Angiosperms also include both dicotyledonous and monocotyledonous plants. Examples of plants include those important in agriculture, particularly in the seed and floriculture industries, such as crop plants such as cereals, flowers, vegetables, and fruits. Specific examples of dicotyledonous plants include species belonging to the Linaceae family (e.g., flax), species belonging to the Fabaceae family (e.g., soybean, peanut, pea, kidney bean, adzuki bean, broad bean, sweet pea), species belonging to the Solanaceae family (e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, petunia), species belonging to the Rosaceae family (e.g., strawberry, rose, apple, pear, peach, loquat, almond, plum, plum, cherry), and species belonging to the Orchidaceae family (e.g., Cymbidium, Phalaenopsis, Examples of monocotyledonous plants include species belonging to the family Liliaceae (e.g., lilies, tulips, hyacinths, muscari, leeks, onions, garlic), family Rutaceae (e.g., mandarins, oranges, grapefruits, lemons, and yuzu), family Vitaceae (e.g., grapes), family Asteraceae (e.g., lettuce, chrysanthemums, dahlias, marigolds, and sunflowers), family Caryophyllaceae (e.g., carnations and baby's breath), and family Theaceae (e.g., camellia and tea plants). Furthermore, examples of monocotyledonous plants include species belonging to the family Poaceae (e.g., rice, wheat, barley, corn, sugarcane, sorghum, and sorghum).

[0050] In one embodiment, this process involves culturing the AM fungus and the second plant in the presence of a porous support to allow the AM fungus to infect the second plant. In this embodiment, the porous support provides a scaffold for the AM fungus' hyphae, thereby increasing the efficiency of infection of the second plant by the AM fungus.

[0051] In another embodiment, this step involves infecting a second plant with the AM fungus produced by the method of the first aspect. The infection method in this embodiment is not limited, and for example, the second plant may be infected with the AM fungus by culturing it in the presence of a porous support to which mycelia and / or spores obtained after the culturing step in the method of the first aspect are attached. Alternatively, the second plant may be infected with the AM fungus by culturing it in soil or a hydroponic solution to which the AM fungus grown by the method of the first aspect has been added, optionally in the presence of a porous support.

[0052] In this process, the roots of the second plant infected with AM fungi can form arbuscules.

[0053] (Cultivation process) In this embodiment, the "cultivation step" refers to cultivating the second plant infected with AM fungi after the infection step. The cultivation conditions in this step are not particularly limited as long as the second plant infected with AM fungi can grow. The cultivation can be performed using a method similar to a general cultivation method depending on the type of second plant. The cultivation temperature is typically 10°C to 40°C, 20°C to 35°C, or 25°C to 30°C. The cultivation period is, for example, 1 day to 3 years, 1 week to 2 years, 2 weeks to 1 year, 1 month to 6 months, or 2 months to 4 months. The cultivation method may be, for example, soil cultivation or hydroponic cultivation. Soil cultivation may be, for example, outdoor cultivation or greenhouse cultivation. Furthermore, cultivation conditions such as light-dark period (light exposure time and dark period), temperature, humidity, and other weather conditions, and growth period may be those known in the art depending on the type of second plant. In this step, a porous support may be used for cultivation to promote the growth of the second plant infected with AM fungi.

[0054] 2-3.Effects According to the method for producing AM fungus-infected plants of the present invention, by culturing the AM fungus and a second plant in the presence of a porous support, the AM fungus can be efficiently infected into the second plant, thereby promoting the growth of the AM fungus-infected plant. Conventional methods have had the problem of not fully achieving the expected effects when plants are cultivated in soil to which fertilizer containing arbuscular mycorrhizal fungi has been added. However, the method of the present invention promotes plant growth through the action of the AM fungus by carrying out the infection step and / or cultivation step in the presence of a porous support, thereby also reducing the amount of chemical fertilizer used.

[0055] 3. Plant cultivation method Overview A third aspect of the present invention is a method for cultivating a plant (hereinafter sometimes abbreviated as "plant cultivation method").

[0056] 3-2. Method The plant cultivation method of this embodiment includes a cultivation step as an essential step.

[0057] (Cultivation process) In this embodiment, the "cultivation step" refers to a step of cultivating a second plant together with the AM fungus produced by the method of the first embodiment. For example, the second plant can be cultivated in the presence of a porous support to which mycelia and / or spores obtained after the culturing step in the method of the first embodiment are attached. Alternatively, the second plant may be cultivated in soil, hydroponic solution, or liquid medium to which mycelia and / or spores of the AM fungus produced by the method of the first embodiment (e.g., spores recovered in the spore recovery step) have been added in the addition step. Furthermore, a second plant infected with an AM fungus in an infection step prior to this step may be cultivated in this step. Specific cultivation conditions are similar to those for the cultivation step of the second embodiment. Cultivation in this step may be carried out in the presence of a porous support. This is because the attachment of AM fungal mycelia to the porous support provides a foothold for the AM fungus, thereby promoting the growth of the second plant.

[0058] 4. Porous Support and AM Fungal Hyphae and / or Spores A fourth aspect of the present invention is a porous support having AM fungal hyphae and / or spores attached thereto, and the AM fungal hyphae and / or spores attached to the porous support. In this aspect, the AM fungal hyphae and / or spores are attached to the porous support, preferably penetrating the pores of the porous support and adhering to the interior of the pores. The spores attached to the porous support can germinate to form hyphae attached to the porous support.

[0059] According to the mycelia and / or spores of this embodiment or the porous support of this embodiment, there is further provided a cultivation container comprising a porous support to which mycelia and / or spores are attached. The cultivation container may further comprise soil or a hydroponic solution.

[0060] By cultivating or cultivating the mycelia and / or spores of this embodiment, or the porous support of this embodiment together with the second plant, the growth of the second plant can be promoted.

[0061] 5. Plant Cultivation Kit A fifth aspect of the present invention is a cultivation set for plants. The cultivation set of this aspect includes a porous support to which AM fungal hyphae and / or spores are attached, a second plant infected with AM fungi, and soil or a hydroponic solution for cultivating the second plant. The cultivation set of this aspect may further include a cultivation container and / or fertilizer (e.g., solid fertilizer or liquid fertilizer).

[0062] In one embodiment, the second plant included in the cultivation set of this aspect is a seed or seedling. The seed of the second plant may be, for example, a seed from which a root or a shoot has emerged. The seedling of the second plant may be, for example, a seedling from which only cotyledons have emerged, a seedling at the three-leaf stage or higher, a small seedling or grandchild seedling (e.g., a runner seedling) propagated from a parent plant, or a clone seedling propagated by cuttings or the like. In the cultivation set of this aspect, the seedling is preferably infected with AM fungi. [Example]

[0063] Example 1: Hydroball-based liquid culture method for AM fungi (the purpose) We will establish a new cultivation method for arbuscular mycorrhiza fungi (Rhizophagus diaphranum) (hereafter abbreviated as "AM fungi").

[0064] (Methods and Results) Linum hairy roots and AM fungi were cultured in liquid culture with or without Hydroballs. Specifically, 80 mL of sterile liquid medium (composition: 1 / 10 MS (Murashige and Skoog medium) + 1% sucrose + 1% KM-Vitamin (Kao and Michaeluk vitamin solution)) was placed in a transparent plastic container (237 mL capacity), and 1.5 L of sterilized medium-sized culture balls (Kaneko Seeds, product number D2310; hereafter referred to as "Hydroballs (I)") were immersed in the medium. Hydroballs (I) are spherical or cylindrical porous supports made of expanded bricks, approximately 0.5 cm to 1.0 cm in diameter and 0.5 cm to 2.0 cm in length. A portion of modified Strullus-Romand (MSR) agar medium (round petri dish) containing flax hairy roots (Linum sp. L. strain flax NM) that had been cultured for three months with AM fungus (Rhizophagus diaphanum (MUCL 49416)) was added to liquid medium and cultured for three months in a sterile environment at 25°C in the dark. A similar culture was also performed without the hydroball (I) immersion as a control.

[0065] Figure 1 shows the growth of flax hairy roots after three months of culture in the presence and absence of Hydroballs (I). Figure 2 shows photographs of flax hairy roots removed from the liquid medium after culture and taken under a stereomicroscope. High-density spore proliferation was induced locally in flax hairy roots cultured in the presence of Hydroballs (I). On the other hand, almost no spore proliferation was observed in flax hairy roots cultured in the absence of Hydroballs (I).

[0066] To quantify the amount of spore growth after cultivation, the hairy roots were removed from the liquid medium, crushed, and the spores were collected using a wet sieve (45 μm diameter). The number of spores was then counted under a microscope. The results are shown in Figures 3 and 4. When cultivated in the presence of Hydroballs (I), spores proliferated more than fourfold compared to cultivation without Hydroballs (I), resulting in the production of 1.4 million spores per liter of culture medium. A significant difference was detected between the two groups when culturing with and without Hydroballs (I) (N = 10, Welch's t-test, p < 0.0001).

[0067] The above results show that the spore production per liter of culture medium is approximately five times higher than that of the conventional method disclosed in JP-A-2016-528906. Furthermore, since the culture period in this example was three months, the spore production per month in this example was 486,000 spores / L. On the other hand, since the culture period in the conventional method disclosed in JP-A-2016-528906 was five months, the spore production per month in this example was approximately eight times higher. Therefore, the method of the present invention significantly shortened the time required for spore production and significantly increased the spore production.

[0068] Example 2: Analysis of spores obtained by liquid culture method based on Hydroball (I) (the purpose) The germination ability of the spores obtained in the presence of Hydroball (I) in Example 1 is examined.

[0069] (Methods and Results) The spores grown in the presence of Hydroball (I) in Example 1 were collected and cultured together with flax hairy roots on an agar medium at 25°C in the dark for 35 days. The appearance of the spores after culture is shown in Figure 5. These results confirmed that the spores had the ability to germinate.

[0070] <Example 3: Examination of various supports> (the purpose) Linum hairy roots and AM fungi are cultured in liquid in the presence of various supports other than the hydroball (I) used in Example 1, and the effects are examined.

[0071] (Methods and Results) In this example, instead of the Hydroball (I) used in Example 1, Hydroball S size (Omiya Green Service Co., Ltd.; hereafter referred to as "Hydroball (II)"), Jiffy Seven PLA (a soil pot ready for planting, Jiffy Seven (Sakata Seed)), rice husks (rice husks for home gardening (Takasho Corporation)), or Mikawa silica sand (Mikawa silica sand No. 5 (Mikawa Silica Sand Co., Ltd.)) were used. Hydroball (II) is a porous support made of expanded brick, with a spherical shape with a diameter of approximately 0.2 cm to approximately 0.6 cm or a cylindrical shape with a length of approximately 0.2 cm to approximately 0.8 cm. The culture conditions, except for the type of support, were the same as in Example 1, and flax hairy roots and AM fungi were cultured in liquid for 3 months. The growth results of flax hairy roots and spores are shown in Table 1 below.

[0072] [Table 1]

[0073] As shown in Table 1 above, Hydroball (II) was found to induce the growth of flax hairy roots and spores, similar to Hydroball (I). In contrast, when Jiffy Seven, rice husks, and Mikawa silica sand were used as supports, no growth of flax hairy roots or spores was observed.

[0074] <Example 4: Observation of mycelia during liquid culture> (the purpose) The state of mycelia during liquid culture in Example 1 was observed.

[0075] (Methods and Results) The hydroballs (I) and flax hairy roots in liquid culture in Example 1 were removed from the liquid medium, and the appearance of the mycelia was photographed under a stereomicroscope. The photographs are shown in Figure 6. As shown in Figure 6, mycelia were observed adhering from the flax hairy roots to the surface of the hydroballs (I). Furthermore, as a result of detailed observation of the mycelia on the surface of the hydroballs (I), it was observed that the mycelia had penetrated into the pores of the hydroballs (I). This result suggests that the mycelia of the AM fungus adhere to the surface of the hydroballs (I) and penetrate into the pores, forming a scaffold.

[0076] Example 5: Microscopic observation of various support surfaces (the purpose) The surface structures of the various supports used in the above examples are observed under a microscope.

[0077] (Methods and Results) The surface structures of Hydroball (I), Hydroball (II), Jiffy Seven PLA, rice husks, and Mikawa silica sand were observed under a microscope. The results are shown in Figures 7 to 11. It was found that pores ranging in size from several tens of μm to approximately 200 μm were distributed on the surfaces of Hydroball (I) and Hydroball (II) (Figures 7 and 8). In contrast, no pores were observed on the surfaces of Jiffy Seven PLA, rice husks, and Mikawa silica sand (Figures 9 to 11). Based on these results and those of Examples 3 and 4, it was suggested that the ability of mycelia to use the pores on the support surface as a foothold is important for the growth of flax hairy roots and AM fungi.

[0078] <Example 6: Analysis of surface structure based on mercury intrusion porosimetry> (the purpose) The cumulative pore volume, cumulative pore specific surface area, and average pore diameter of Hydroball (I) and Hydroball (II) are analyzed by mercury intrusion porosimetry.

[0079] (Methods and Results) The analysis of Hydroball (I) and Hydroball (II) was outsourced to Sumika Chemical Analysis Center and analyzed by mercury intrusion porosimetry. Specifically, after pretreatment by constant temperature drying at 120°C for 4 hours, measurements were performed using an Autopore V9620 (manufactured by Micromeritics) with a surface tension of mercury of 480 dynes / cm and a contact angle of mercury with the sample of 140 degrees. The measurement results are shown in Table 2 below.

[0080] [Table 2]

[0081] Furthermore, elemental analysis of Hydroball (I) and Hydroball (II) revealed that the main components of both were silicon oxide and aluminum oxide.

Claims

1. 1. A method for producing arbuscular mycorrhizal fungi, comprising: a culturing step in which the arbuscular mycorrhizal fungus and the first plant to be infected with the arbuscular mycorrhizal fungus are cultured in the presence of a porous support to allow the arbuscular mycorrhizal fungus to grow. The method comprising:

2. The method according to claim 1 , wherein, during the culturing step, the hyphae of the arbuscular mycorrhizal fungi attach to the porous support and / or penetrate into the pores of the porous support.

3. The method of claim 1 , wherein the culturing step cultures the roots of the first plant.

4. The method of claim 3 , wherein the roots are hairy roots.

5. The method of claim 1 , wherein the first plant is other than the Brassicaceae and Chenopodiaceae families.

6. The method of claim 1 , wherein the culture is an axenic culture.

7. The method of claim 1 , wherein the culture is a liquid culture.

8. The method according to claim 7 , wherein the specific gravity of the porous support is greater than the specific gravity of the culture medium used for the liquid culture.

9. The method according to claim 1, wherein the average pore diameter of the porous support is 100 nm or more and 1 μm or less.

10. The method according to any one of claims 1 to 9, wherein the porous support is made of expanded brick.

11. a spore recovery step of recovering the arbuscular mycorrhizal fungal spores obtained after the culture step. The method of claim 1 further comprising:

12. 1. A method for producing a plant infected with arbuscular mycorrhizal fungi, comprising: an infection step in which an arbuscular mycorrhizal fungus and a plant to be infected with the arbuscular mycorrhizal fungus are cultured in the presence of a porous support to infect the plant with the arbuscular mycorrhizal fungus; The method comprising:

13. 1. A method for producing a plant infected with arbuscular mycorrhizal fungi, comprising: an infection step in which the arbuscular mycorrhizal fungus produced by the method of claim 1 is infected into a second plant; The method comprising:

14. A method for cultivating a plant, comprising: a cultivation step of cultivating a second plant to be infected with the arbuscular mycorrhizal fungus together with the arbuscular mycorrhizal fungus produced by the method of claim 1; The method comprising:

15. The method according to claim 14, wherein the cultivation step is soil cultivation or hydroponic cultivation.

16. an addition step of adding the arbuscular mycorrhizal fungi to the soil used for the soil cultivation or the hydroponic solution used for the hydroponic cultivation before the cultivation step; 16. The method of claim 15, further comprising:

17. an infection step in which the arbuscular mycorrhizal fungus is infected into the second plant before the cultivation step; 15. The method of claim 14, further comprising:

18. The method according to any one of claims 13 to 17, wherein the second plant is other than the Brassicaceae and Chenopodiaceae families.

19. A porous support to which hyphae and / or spores of arbuscular mycorrhizal fungi are attached.

20. Arbuscular mycorrhizal fungal hyphae and / or spores attached to a porous support.

21. A plant cultivation set, A cultivation set comprising the porous support according to claim 19, the arbuscular mycorrhizal fungus, a plant infected with the arbuscular mycorrhizal fungus, and soil or a hydroponic solution for cultivating the plant.

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